Islet Cells from Human Embryonic Stem Cells

ABSTRACT

This disclosure provides a system for producing pancreatic islet cells from embryonic stem cells. Differentiation is initiated towards endoderm cells, and focused using reagents that promote emergence of islet precursors and mature insulin-secreting cells. High quality populations of islet cells can be produced in commercial quantities for use in research, drug screening, or regenerative medicine.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/262,633 filed Oct. 31, 2005, and a divisional of U.S. patentapplication Ser. No. 10/313,739, filed Dec. 6, 2002 (pending), whichclaims priority to U.S. provisional application 60/338,885, filed Dec.7, 2001 (expired). The priority application is hereby incorporatedherein by reference in its entirety, as is International PatentPublication WO 03/050249.

TECHNICAL FIELD

This invention relates generally to the fields of cell biology,embryonic stem cells, and cell differentiation. More specifically, thisinvention provides differentiated cells with pancreatic endocrinefunction.

BACKGROUND

The American Diabetes Association estimates that there are currently 5million people in the United States with confirmed diabetes, and over 10million at risk.

The cost of this disease and its sequelae to the American economy isstaggering. Care of diabetics consumes a total of $98 billion per year,accounting for one of every seven healthcare dollars spent in the U.S.There are 24,000 new cases of diabetes-caused blindness caused bydiabetes each year. Diabetes is the leading cause of kidney failure,contributing about 40% of new dialysis patients. Diabetes is also themost frequent cause of lower limb amputation, with 56,000 limbs lost todiabetes each year. The per capita health care costs incurred perdiabetic person is $10,071 annually, compared with $2,669 fornon-diabetics.

Type I diabetes mellitus (also known as insulin-dependent diabetes) is asevere condition accounting for 5-10% all diabetics. The pathologyarises because the patient's insulin-secreting beta cells in thepancreas have been eliminated by an autoimmune reaction. Under currentpractice, the condition is managed by regular injection of insulin,constant attention to diet, and continuous monitoring of blood glucoselevels to adjust the insulin dosing. It is estimated that the market forrecombinant insulin will reach $4 billion by 2005. Of course, theavailability of insulin is life-saving for Type I diabetics. But thereis no question that the daily regimen of administration and monitoringthat diabetics must adhere to is troublesome to the end user, and notuniversally effective.

For this reason, there are several clinical tests underway to transplantdiabetics with islet cells isolated from donor pancreas. This has beenmade possible by recent advances in the isolation and culture of isletcells. U.S. Pat. No. 4,797,213 described separation of islets ofLangerhans. U.S. Pat. No. 4,439,521 reports a method for producingself-reproducing pancreatic islet-like structures. U.S. Pat. No.5,919,703 reports preparation and storage of pancreatic islets. U.S.Pat. No. 5,888,816 reports cell culture techniques for pancreatic cellsusing hypothalamus and pituitary extracts. WO 00/72885 reports methodsof inducing regulated pancreatic hormone production in non-pancreaticislet tissues. WO 00/78929 reports methods of making pancreatic isletcells. Kim et al. (Genes Dev. 15:111, 2001) review the intercellularsignals regulating pancreas development and function. Yamaoka et al.(Int. J. Mol. Med. 3:247, 1999) review the development of pancreaticislets, and the putative role of factors such as Sonic hedgehog andactivin, transcriptional factors like PDX1 and Isl1, growth factors likeEGF and HGF, hormones like insulin and growth hormone, and cell adhesionmolecules such as N-CAM and cadherins.

Peck et al. (Ann. Med. 33:186, 2001) propose that pancreatic stem cellsbe used as building blocks for better surrogate islets for treating TypeI diabetes. WO 00/47721 reports methods of inducing insulin positiveprogenitor cells. WO 01/39784 reports pancreatic stem cells isolatedfrom islet cells that are nestin-positive. WO 01/77300 reports humanpancreatic epithelial progenitors that are proposed to have the capacityto differentiate into acinar, ductal, and islet cells. Deutsch et al.(Development 128:871, 2001) describe a bipotential precursor populationfor pancreas and liver within the embryonic endoderm. Zulewski et al.(Diabetes 50:521, 2001) describe multipotential nestin-positive stemcells isolated from adult pancreatic islets that differentiate intoendocrine, exocrine, and hepatic phenotypes. U.S. Pat. No. 6,326,201(Curis Inc.) reports pancreatic progenitor cells made by dissociatingand culturing cells from pancreatic duct. The present clinicalexperience of islet cell transplantation is reviewed by Bretzel et al.(Exp. Clin. Endocrinol. Diabetes 190 (Suppl. 2):S384, 2001) andOberholzer et al. (Ann. N.Y. Acad. Sci. 875:189, 1999). The currentclinical trials typically involve infusing cells from at least twopancreas donors. Even if this treatment proves to be successful, therewill be insufficient material available from current sources to treatall the eligible Type I diabetic patients.

Developmental work has been done in several institutions to capitalizeon the promise of pluripotent stem cells from the embryo todifferentiate into other cell types. Cells bearing features of the isletcell lineage have reportedly been derived from embryonic cells of themouse. For example, Lumelsky et al. (Science 292:1389, 2001) reportdifferentiation of mouse embryonic stem cells to insulin-secretingstructures similar to pancreatic islets. Soria et al. (Diabetes 49:157,2000) report that insulin-secreting cells derived from mouse embryonicstem cells normalize glycemia in streptozotocin-induced diabetic mice.

Regrettably, the mouse model of embryonic stem cell development is itsown peculiar case, and does not yield strategies for differentiationthat are applicable to other species. In fact, pluripotent stem cellshave been reproducibly isolated from very few other mammalian species.Only recently did Thomson et al. isolate embryonic stem cells from humanblastocysts (Science 282:114, 1998). Concurrently, Gearhart andcoworkers derived human embryonic germ (hEG) cell lines from fetalgonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726,1998). Unlike mouse embryonic stem cells, which can be kept fromdifferentiation simply by culturing with Leukemia Inhibitory Factor(LIF), human embryonic stem cells must be maintained under very specialconditions (U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616).Accordingly, it is necessary to develop completely new paradigms todifferentiate human pluripotent cells into fully functionaldifferentiated cell types.

Jacobson et al. (Transplant. Proc. 33:674, 2001) reporteddifferentiation of intestinal and pancreatic endoderm from rhesusembryonic stem cells. Assady et al. (Diabetes 50:1691, 2001) identifiedinsulin production by human embryonic stem cells differentiated toembryoid bodies, by immunohistochemistry and enzyme-linked immunoassayof the culture medium. Of course, embryoid bodies contain an enormousvariety of different cell types (WO 01/51616), and Assady made noattempt to isolate the insulin-secreting cells or determinedifferentiation conditions that would produce enriched populations.

For embryonic stem cell derived islet cells to become a commerciallyviable proposition, there is a need to develop new procedures thatprovide for populations of islet cells of high purity.

SUMMARY

This invention provides a system for efficient production of primatecells that have differentiated from pluripotent cells into cells of theislet cell lineage. Populations of cells are described that areconsiderably enriched for islet progenitor cells. In turn, the isletprogenitors can be further differentiated into colonies comprising cellsthat secrete insulin, glucagon, somatostatin, or a combination of allthree.

Accordingly, one embodiment of the invention is a cell populationobtained by differentiating primate pluripotent stem (pPS) cells (suchas embryonic stem cells), in which at least 5% of the cells secrete atleast one of the four islet cell proteins from an endogenous gene: thehormones insulin, glucagon, somatostatin, and the apparently inertproduct known as pancreatic polypeptide. The cells may be in clusters,comprising cells secreting each of the three endocrines, and may beprocessed to contain a minimum proportion of other tissue types. Thecells of this invention may be identified by phenotypic markers listedlater on in this disclosure. Functional efficacy can be confirmed by theability to improve fasting glucose levels when administered to ahyperglycemic subject.

Another embodiment of the invention is a differentiated cell populationcapable of self-renewal, and capable of forming progeny that are matureislet cells. This means that the cells are no longer pluripotent, butretain the ability to form islet cells upon proliferation. Theproportion of undifferentiated pluripotent cells in the population ispreferably minimized, and any residual undifferentiated cells are notthe cells responsible for forming the islet cells upon furtherproliferation. Optionally, replication capacity of the stem cells can beimproved by increasing telomerase activity.

Another embodiment of the invention is a method for obtainingpolypeptide-secreting cells, in which differentiation of pPS cells isinitiated, for example, by forming embryoid bodies or other formcomprising early ectoderm. The cells are then cultured in a mixture ofdifferentiation factors, such as TGF-β antagonists like Noggin, activinA, n-butyrate, or combinations of the other factors listed below. Inaddition or as an alternative, the cells can be genetically altered tocause expression of a pancreatic transcription factor such as Neurogenin3.

A further embodiment of the invention is a method of screening acompound for its ability to modulate islet cell function, using a cellcomposition of the invention.

Another embodiment of this invention is a method for making insulin,glucagon, or somatostatin by growing the islet cells of this invention.Also included are pharmaceutical compositions and devices containing thecells of this invention. The cells, compositions, and devices of thisinvention are useful for reconstituting islet cell function in asubject, especially but not limited to the treatment of Type I diabetes.

These and other embodiments of the invention are further elucidated inthe description that follows.

DRAWINGS

FIG. 1 shows hES-derived hepatocytes during the differentiation andmaturation process (10×, 40×). Row A shows cells 4 days after culture inmedium containing 5 mM sodium n-butyrate. More than 80% of cells in theculture contain large nuclei and granular cytoplasm. The cells wereswitched to specialized hepatocyte culture medium for 2-4 days (Rows Band C). Multinucleated polygonal cells are common. The ES-derivedhepatocytes share morphological features with freshly isolated humanadult hepatocytes (Row D) and fetal hepatocytes (Row E).

FIG. 2 shows hES-derived cells expressing insulin. The differentiationstrategy is based on the hypothesis that hepatocytes and pancreaticcells have a common early progenitor. Initial differentiation wassimilar to that of hepatocytes, except that cyclopamine was included inthe culture medium. The cells were next cultured with the isletdifferentiation factors activin A, nicotinamide, cyclopamine, and a lowconcentration of n-butyrate. Finally, the cells were cultured for 11days with activin A, betacellulin, nicotinamide, and IGF-1 to promoteoutgrowth of islet cells. DAPI stained cell nuclei are shown in theupper panel. The corresponding field in the lower panel shows diffusered antibody staining for insulin.

FIG. 3 shows results of a protocol in which hES cells are being directedalong the pathway of islet cell ontogeny in a step-wise fashion.Expression of the three gut endoderm markers was assayed at the mRNAlevel, and normalized to expression levels in pancreas. Expression inundifferentiated or nonspecifically differentiated cells was low, but acombination of n-Butyrate and Activin A caused differentiation orselection of cells having characteristics for gut endoderm.

FIG. 4 shows results of a protocol in which hES cells were put inlong-term aggregate culture in a transition medium, and then in a mediumcontaining mitogens and the islet cell differentiation factor Noggin.The top and middle panels show staining for insulin c-peptide at low andhigh magnification, indicating a cluster of mature pancreatic betacells. The bottom panel shows staining for somatostatin, a marker ofdelta cells.

FIG. 5 shows cells in an islet cell differentiation paradigm, 9 daysafter transfecting with a vector containing the transcription regulatorgene Neurogenin 3. The cells show a high level of antibody-detectableglucagon expression.

FIGS. 6(A) and (B) shows mRNA expression levels in the Neurogenin3transduced cells (solid bars), compared with the negative control(hatched bars). The transfected gene causes upregulation downstreamgenes NeuroD1 and Nkx2.2 as early as day 2, leading to enhancedexpression of insulin and glucagon.

DETAILED DESCRIPTION

This invention solves the problem of generating large populations ofhuman islet cells by showing how to efficiently differentiate them frompluripotent stem cells.

It has been discovered that stem cells can be coaxed along the isletcell differentiation pathway by initiating differentiation towards theendodermal lineage, and focusing the differentiation process byculturing in the presence of factors that facilitate outgrowth of isletcells. This invention provides a system for producing a population ofcells enriched for multipotent islet cell progenitors, capable offorming mature islets. If desired, the differentiation process cancontinue in order to maintain mature endocrine-secreting cells.

As an aid to optimizing the differentiation process, this disclosureprovides a strategy of dividing the differentiation pathway into aseries of sequential stages. In this way, factors effective in pushingthe cells along each segment of the differentiation pathway can beidentified.

In the illustration described in Example 5, the differentiation processproceeded as follows. For Stage 1, undifferentiated human embryonic stemcells from feeder free culture were differentiated so as to form a mixedcell aggregate containing endoderm cells in suspension culture. Retinoicacid was used as the initial differentiation agent, in combination withenrichment factors selenium and T3. For Stage 2, differentiation topancreas progenitor cells was effected by culturing in a mediumcontaining Noggin (200 ng/ml), EGF (20 ng/ml) and bFGF (2 ng/ml). ForStage 3, differentiation to end-stage islet cells was induced bywithdrawing the Noggin, EGF and bFGF, and instead culturing the cellswith 10 mM nicotinamide. As shown in FIG. 4, clusters of cells wereobtained synthesizing antibody-detectable levels of c-peptide of insulinand somatostatin.

Methods to efficiently produce islet stem cells from pPS cells areimportant, because pPS cells can be caused to proliferate indefinitely.This invention provides a system that can be used to generate unboundedquantities of islet progenitors—and progeny that are committed to formmature islet cells.

The disclosure that follows provides further information on theproduction and testing of islet cells of this invention. It alsoprovides extensive illustrations of how these cells can be used inresearch, pharmaceutical development, and the therapeutic management ofconditions related to islet cell dysfunction.

DEFINITIONS

For purposes of this disclosure, the term “islet cell” refers toterminally differentiated pancreatic endocrine cells, and any precursorcell that is committed to form progeny normally classified as pancreaticendocrine. The cell expresses some of the accepted morphologicalfeatures and phenotypic markers (exemplified below) that arecharacteristic of the islet cell lineage. Mature alpha cells secreteglucagon; mature beta cells secrete insulin; mature delta cells secretesomatostatin; PP cells secrete pancreatic polypeptide.

An “islet progenitor”, “islet precursor” or “islet stem cell” is anislet cell that does not substantially secrete endocrines, but has thecapability to proliferate and generate terminally differentiated cells.It may also have the capability to self-renew. Early islet progenitorsare multipotent, which means that they are capable of forming at leasttwo and potentially all four mature islet cell types.

A “pancreas progenitor”, precursor, or stem cell is capable of formingboth pancreatic endocrine and pancreatic exocrine cells.

In the context of cell ontogeny, the adjective “differentiated” is arelative term. A “differentiated cell” is a cell that has progressedfurther down the developmental pathway than the cell it is beingcompared with. It is a hypothesis of this invention that pluripotentembryonic stem cells in the course of normal ontogeny differentiatefirst to an endoderm cell that is capable of forming pancreas cells andother endoderm cell types. Further differentiation leads to thepancreatic pathway, where ˜98% of the cells become exocrine, ductular,or matrix cells, and ˜2% become endocrine cells. Early endocrine cellsare islet progenitors, which then differentiate further into functionalendocrine cells specializing in secretion of insulin, glucagon,somatostatin, or pancreatic polypeptide.

A “differentiation agent”, as used in this disclosure, refers to one ofa collection of compounds that are used in culture systems of thisinvention to produce differentiated cells of the islet lineage(including precursor cells and terminally differentiated cells). Nolimitation is intended as to the mode of action of the compound. Forexample, the agent may assist the differentiation process by inducing orassisting a change in phenotype, promoting growth of cells with aparticular phenotype or retarding the growth of others. It may also actas an inhibitor to other factors that may be in the medium orsynthesized by the cell population that would otherwise directdifferentiation down the pathway to an unwanted cell type.

Prototype “primate Pluripotent Stem cells” (pPS cells) are pluripotentcells derived from pre-embryonic, embryonic, or fetal tissue at any timeafter fertilization, and have the characteristic of being capable underappropriate conditions of producing progeny of several different celltypes that are derivatives of all of the three germinal layers(endoderm, mesoderm, and ectoderm), according to a standard art-acceptedtest, such as the ability to form a teratoma in 8-12 week old SCID mice.The term includes both established lines of stem cells of various kinds,and cells obtained from primary tissue that are pluripotent in themanner described.

Included in the definition of pPS cells are embryonic cells of varioustypes, exemplified by human embryonic stem (hES) cells, described byThomson et al. (Science 282:1145, 1998); embryonic stem cells from otherprimates, such as Rhesus stem cells (Thomson et al., Proc. Natl. Acad.Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol.Reprod. 55:254, 1996) and human embryonic germ (hEG) cells (Shamblott etal., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Other types ofpluripotent cells are also included in the term. Any cells of primateorigin that are capable of producing progeny that are derivatives of allthree germinal layers are included, regardless of whether they werederived from embryonic tissue, fetal tissue, or other sources. The pPScells are preferably not derived from a malignant source. It isdesirable (but not always necessary) that the cells be karyotypicallynormal.

pPS cell cultures are described as “undifferentiated” when a substantialproportion of stem cells and their derivatives in the population displaymorphological characteristics of undifferentiated cells, clearlydistinguishing them from differentiated cells of embryo or adult origin.Undifferentiated pPS cells are easily recognized by those skilled in theart, and typically appear in the two dimensions of a microscopic view incolonies of cells with high nuclear/cytoplasmic ratios and prominentnucleoli. It is understood that colonies of undifferentiated cellswithin the population will often be surrounded by neighboring cells thatare differentiated.

“Feeder cells” are cells of one type that are co-cultured with cells ofanother type, to provide an environment in which the cells of the secondtype can grow. Certain types of pPS cells can be supported by primarymouse embryonic fibroblasts, immortalized mouse embryonic fibroblasts,or human fibroblast-like cells differentiated from hES cell. pPS cellpopulations are said to be “essentially free” of feeder cells if thecells have been grown through at least one round after splitting inwhich fresh feeder cells are not added to support growth of the pPScells.

The term “embryoid bodies” is a term of art synonymous with “aggregatebodies”, referring to aggregates of differentiated and undifferentiatedcells that appear when pPS cells overgrow in monolayer cultures, or aremaintained in suspension cultures. Embryoid bodies are a mixture ofdifferent cell types, typically from several germ layers,distinguishable by morphological criteria and cell markers detectable byimmunocytochemistry.

A “growth environment” is an environment in which cells of interest willproliferate, differentiate, or mature in vitro. Features of theenvironment include the medium in which the cells are cultured, anygrowth factors or differentiation factors that may be present, and asupporting structure (such as a substrate on a solid surface) ifpresent.

A cell is said to be “genetically altered” or “transfected” when apolynucleotide has been transferred into the cell by any suitable meansof artificial manipulation, or where the cell is a progeny of theoriginally altered cell that has inherited the polynucleotide.

General Techniques

General methods in molecular genetics and genetic engineering aredescribed in the current editions of Molecular Cloning: A LaboratoryManual, (Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors forMammalian Cells (Miller & Calos eds.); and Current Protocols inMolecular Biology (F. M. Ausubel et al. eds., Wiley & Sons). Cellbiology, protein chemistry, and antibody techniques can be found inCurrent Protocols in Protein Science (J. E. Colligan et al. eds., Wiley& Sons); Current Protocols in Cell Biology (J. S. Bonifacino et al.,Wiley & Sons) and Current protocols in Immunology (J. E. Colligan et al.eds., Wiley & Sons.). Reagents, cloning vectors, and kits for geneticmanipulation referred to in this disclosure are available fromcommercial vendors such as BioRad, Stratagene, Invitrogen, ClonTech, andSigma-Aldrich Co.

Cell culture methods are described generally in the current edition ofCulture of Animal Cells: A Manual of Basic Technique (R. I. Freshneyed., Wiley & Sons); General Techniques of Cell Culture (M. A. Harrison &I. F. Rae, Cambridge Univ. Press), and Embryonic Stem Cells: Methods andProtocols (K. Turksen ed., Humana Press). Tissue culture supplies andreagents are available from commercial vendors such as Gibco/BRL,Nalgene-Nunc International, Sigma Chemical Co., and ICN Biomedicals.

Specialized works relevant to this disclosure include The ComparativePhysiology of the Pancreatic Islet, by J. E. Brinn, Springer-Verlag1988; Pancreatic Islet Cell Regeneration and Growth, by E. J. Vinik etal. eds., Kluwer 1992; and Immunomodulation of Pancreatic Islet(Pancreatic Islet Transplantation, Vol 2), by R. P. Lanza et al eds.,Springer Verlag 1994.

Sources of Stem Cells

This invention can be practiced using stem cells of various types.Amongst the stem cells suitable for use in this invention are primatepluripotent stem (pPS) cells derived from tissue formed after gestation,such as a blastocyst, or fetal or embryonic tissue taken any time duringgestation. Non-limiting examples are primary cultures or establishedlines of embryonic stem cells or embryonic germ cells, as exemplifiedbelow.

The techniques of this invention can also be implemented directly withprimary embryonic or fetal tissue, deriving islet cells directly fromprimary cells that have the potential to give rise to islet cellswithout first establishing an undifferentiated cell line. Under certaincircumstances, the methods of this invention may also be invoked usingmultipotent cells from cord blood, placenta, or certain adult tissues.

Embryonic Stem Cells

Embryonic stem cells can be isolated from blastocysts of members of theprimate species (U.S. Pat. No. 5,843,780; Thomson et al., Proc. Natl.Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells can beprepared from human blastocyst cells using the techniques described byThomson et al. (U.S. Pat. No. 6,200,806; Science 282:1145, 1998; Curr.Top. Dev. Biol. 38:133 ff., 1998) and Reubinoffetal, Nature Biotech.18:399, 2000. Equivalent cell types to hES cells include theirpluripotent derivatives, such as primitive ectoderm-like (EPL) cells, asoutlined in WO 01/51610 (Bresagen).

hES cells can be obtained from human preimplantation embryos.Alternatively, in vitro fertilized (IVF) embryos can be used, orone-cell human embryos can be expanded to the blastocyst stage (Bongsoet al., Hum Reprod 4: 706, 1989). Embryos are cultured to the blastocyststage in G1.2 and G2.2 medium (Gardner et al., Fertil. Steril. 69:84,1998). The zona pellucida is removed from developed blastocysts by briefexposure to pronase (Sigma). The inner cell masses are isolated byimmunosurgery, in which blastocysts are exposed to a 1:50 dilution ofrabbit anti-human spleen cell antiserum for 30 min, then washed for 5min three times in DMEM, and exposed to a 1:5 dilution of Guinea pigcomplement (Gibco) for 3 min (Solter et al., Proc. Natl. Acad. Sci. USA72:5099, 1975). After two further washes in DMEM, lysed trophectodermcells are removed from the intact inner cell mass (ICM) by gentlepipetting, and the ICM plated on mEF feeder layers.

After 9 to 15 days, inner cell mass-derived outgrowths are dissociatedinto clumps, either by exposure to calcium and magnesium-freephosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispaseor trypsin, or by mechanical dissociation with a micropipette; and thenreplated on mEF in fresh medium. Growing colonies havingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and replated. ES-like morphologyis characterized as compact colonies with apparently high nucleus tocytoplasm ratio and prominent nucleoli. Resulting ES cells are thenroutinely split every 1-2 weeks by brief trypsinization, exposure toDulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase(˜200 U/mL; Gibco) or by selection of individual colonies bymicropipette. Clump sizes of about 50 to 100 cells are optimal.

Embryonic Germ Cells

Human Embryonic Germ (hEG) cells can be prepared from primordial germcells present in human fetal material taken about 8-11 weeks after thelast menstrual period. Suitable preparation methods are described inShamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S.Pat. No. 6,090,622.

Briefly, genital ridges processed to form disaggregated cells. EG growthmedium is DMEM, 4500 mg/L D-glucose, 2200 mg/L mM NaHCO₃; 15% ESqualified fetal calf serum (BRL); 2 mM glutamine (BRL); 1 mM sodiumpyruvate (BRL); 1000-2000 U/mL human recombinant leukemia inhibitoryfactor (LIF, Genzyme); 1-2 ng/mL human recombinant bFGF (Genzyme); and10 μM forskolin (in 10% DMSO). Ninety-six well tissue culture plates areprepared with a sub-confluent layer of feeder cells (e.g., STO cells,ATCC No. CRL 1503) cultured for 3 days in modified EG growth medium freeof LIF, bFGF or forskolin, inactivated with 5000 rad γ-irradiation. ˜0.2mL of primary germ cell (PGC) suspension is added to each of the wells.The first passage is done after 7-10 days in EG growth medium,transferring each well to one well of a 24-well culture dish previouslyprepared with irradiated STO mouse fibroblasts. The cells are culturedwith daily replacement of medium until cell morphology consistent withEG cells is observed, typically after 7-30 days or 1-4 passages.

Propagation of pPS Cells in an Undifferentiated State

pPS cells can be propagated continuously in culture, using cultureconditions that promote proliferation without promoting differentiation.Exemplary serum-containing ES medium is made with 80% DMEM (such asKnock-Out DMEM, Gibco), 20% of either defined fetal bovine serum (FBS,Hyclone) or serum replacement (WO 98/30679), 1% non-essential aminoacids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Just before use,human bFGF is added to 4 ng/mL (WO 99/20741, Geron Corp.).Traditionally, ES cells are cultured on a layer of feeder cells,typically fibroblasts derived from embryonic or fetal tissue.

Scientists at Geron have discovered that pPS cells can be maintained inan undifferentiated state even without feeder cells. The environment forfeeder-free cultures includes a suitable culture substrate, particularlyan extracellular matrix such as Matrigel® or laminin. Typically,enzymatic digestion is halted before cells become completely dispersed(say, ˜5 min with collagenase IV). Clumps of ˜10 to 2,000 cells are thenplated directly onto the substrate without further dispersal.

Feeder-free cultures are supported by a nutrient medium containingfactors that support proliferation of the cells without differentiation.Such factors may be introduced into the medium by culturing the mediumwith cells secreting such factors, such as irradiated (˜4,000 rad)primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, orfibroblast-like cells derived from pPS cells. Medium can be conditionedby plating the feeders at a density of ˜5−6×10⁴ cm⁻² in a serum freemedium such as KO DMEM supplemented with 20% serum replacement and 4ng/mL bFGF. Medium that has been conditioned for 1-2 days issupplemented with further bFGF, and used to support pPS cell culture for1-2 days. Features of the feeder-free culture method are furtherdiscussed in International Patent Publication WO 01/51616; and Xu etal., Nat. Biotechnol. 19:971, 2001.

Under the microscope, ES cells appear with high nuclear/cytoplasmicratios, prominent nucleoli, and compact colony formation with poorlydiscernable cell junctions. Primate ES cells express stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science282:1145, 1998). Mouse ES cells can be used as a positive control forSSEA-1, and as a negative control for SSEA-4, Tra-1-60, and Tra-1-81.SSEA-4 is consistently present on human embryonal carcinoma (hEC) cells.Differentiation of pPS cells in vitro results in the loss of SSEA-4,Tra-1-60, and Tra-1-81 expression, and increased expression of SSEA-1,which is also found on undifferentiated hEG cells.

Materials and Procedures for Preparing Islet Cells and their Derivatives

Islet cells of this invention are obtained by culturing,differentiating, or reprogramming stem cells in a special growthenvironment that enriches for cells with the desired phenotype (eitherby outgrowth of the desired cells, or by inhibition or killing of othercell types). These methods are applicable to many types of stem cells,including primate pluripotent stem (pPS) cells described in the previoussection.

Step-Wise Differentiation

It is a hypothesis of the invention that the production and enrichmentof islet cells from pPS cells may be facilitated by forming anearly-stage progenitor that is multipotential for formation ofpancreatic cells and other cell types. In a general sense, this strategyinvolves first forming a cell population enriched for a relevantcommitted common precursor cell, and then further differentiating intomore mature cells that are more and more specialized towards theformation of islets. According to this strategy, differentiation of pPScells towards mature islets is done in several deliberate stages.

One intermediate between undifferentiated pPS cells and mature islets isan immature endoderm cell. Early in ontogeny, endoderm cells are capableof making epithelial cells of the GI tract and respiratory system, andthe key digestive organs (liver and pancreas). Islet cells can begenerated using a two-stage approach. Stage 1 involves obtaining apopulation of common endoderm precursor cells. Stage 2 involves maturingthe endoderm precursors into pancreatic endocrine. As illustrated inExample 3, pPS cells can be initiated along the endoderm differentiationpathway by culturing with the hepatocyte differentiation agentn-butyrate. Further elaboration of the hepatocyte differentiationparadigm is described in International Patent Publication WO 01/81549(Geron Corporation). Sonic Hedgehog is thought to be involved in liverspecification, so including cyclopamine (an inhibitor of Sonic Hedgehog)in the culture medium is thought to help divert the cells toward thepancreatic lineage. Differentiation can then be pushed further in asubsequent step, using the terminal differentiation factor nicotinamide(in the presence of cyclopamine and activin A).

In a further manifestation of this approach, the differentiation pathwayis divided into three stages. pPS cells are first differentiated toendoderm (Stage 1), and then to a second intermediate (Stage 2)—say, thelevel of a committed pancreas precursor (identifiable with the markerPdx1). A further differentiation step (Stage 3) can be performed if theuser wants to obtain mature islets. By way of illustration, toaccomplish Stage I, pPS cells can be differentiated to cells havingmarkers for gut endoderm using a combination of n-butyrate and activin A(Example 4). Alternatively, a heterogeneous population comprisingendodermal cells can be prepared by culturing pPS cells with retinoicacid in the presence of enriching agents (selenium and thyroid hormonessuch as T3) (Example 5). To accomplish Stage 2, the cells can becultured with TGF-β antagonists such as Noggin, in combination withmitogens (a member of the FGF family, possibly in combination with EGFor betacellulin) (Example 5). It may also be helpful to block hedgehogsignaling with cyclopamine. Stage 3 can be accomplished as alreadydescribed, using nicotinamide as the terminal differentiation agent(Example 5). Alternatively, transcription factors can be activated bydirect manipulation that causes progression from Pdx1 positivepancreatic precursors to mature islet cells (Example 6).

This step-wise approach to differentiation is intended as a guide to thereader, and does not limit the invention except where explicitlyindicated. The differentiation pathway can be broken down into even morestages so that step-wise differentiation can be optimized in anincremental fashion. For example, a potential intermediate betweenpancreatic precursors and mature islets are precursor cells committed toform pancreatic endocrine. On the other hand, depending on thecircumstances, effective differentiation agents may be combined to workon cells in different stages at the same time, or to promote a cascadingeffect down the differentiation pathway.

The desired end-stage cell population will depend in part on itsintended use. For example, committed islet progenitor cells may be ofparticular value for therapy of generalized islet insufficiency, andstudying islet differentiation in vitro. Earlier progenitors may havegreater capacity for self-renewal. Mature cell populations showinghigh-level synthesis of insulin or other endocrines where immediateproduction of the hormone is required. The differentiation process istailored accordingly, stopping the process at a stage that yields thedesired level of maturation.

The following sections provide tissue culture and gene transfectiontechniques that are effective in promoting differentiation in the mannerdescribed.

Initiating the Differentiation Process

There are two approaches to begin differentiation of pPS cells towardsendodermal cells. One is to plate the cells onto a new substrate, orexchange the medium to remove extracellular matrix or soluble factorsthat inhibit differentiation. This is sometimes referred to as the“direct differentiation method”, and is described in general terms inInternational patent publication WO 01/51616, and U.S. PatentPublication 20020019046. It is usually preferable in the directdifferentiation method to begin with a feeder-free culture of pPS cells,so as to avoid potential complications in the differentiation processcaused by residual feeder cells. Example 4 provides an illustration inwhich gut endoderm is produced by direct differentiation by introducingmedium containing early differentiation factors.

The other approach is to put undifferentiated pPS cells in suspensionculture, which will frequently cause them to form embryoid bodies oraggregates. For example, pPS cells are harvested by brief collagenasedigestion, dissociated into clusters, and passaged in non-adherent cellculture plates. The aggregates are fed every few days, and thenharvested after a suitable period, typically 4-8 days (Examples 1 & 5).In some instances, differentiation is enhanced by other factors in themedium: for example, retinoic acid (Example 5) or dimethyl sulfoxide(DMSO). Depending on the conditions, aggregates will generally start byforming a heterogeneous population of cell types, including asubstantial frequency of endoderm cells. The embryoid bodies can then bedispersed and replated for the next stage in the differentiationprocess, on substrates such as laminin or fibronectin; or passaged insuspension culture, using non-adherent plates and a suitable medium.

Direct differentiation or differentiation in aggregates can be monitoredfor the presence of endoderm cells using the markers listed below. Oncea sufficient proportion of endoderm is obtained, cells are replated orotherwise manipulated to begin Stage II. In certain circumstances,differentiation or maintenance of islet cells may be enhanced if thecells are kept in micromass clusters (for example, 50 to 5,000 cells),so that alpha, beta, and delta cells can interact directly.

Once the common progenitor cells are made in this manner, they can becultured with specific differentiation factors and/or induced withislet-specific genes or promoters as described in the sections thatfollow.

Driving Differentiation Further Towards Islet Cells Using SolubleFactors

In order to drive the culture further down the stages of the isletpathway, pPS cells or their differentiated progeny may be cultured in acocktail of islet differentiation factors. Alone or in combination, eachof the factors may increase the frequency of conversion to the desiredcell type, cause outgrowth of cells with a islet phenotype, inhibitgrowth of other cell types, or enrich for islet cells in anotherfashion. It is not necessary to understand the mechanism resulting inislet cells being enriched in order to practice the invention. Whatfollows is a non-limiting list of candidate differentiation factors.

TABLE 1 Factors for Differentiation of Islet Cells from pPS CellsInitial working Factor Compound type or family Proposed functionconcentration Cyclopamine steroidal alkaloid inhibitor of hedgehogsignaling; may 10 μM also act as inhibitor of cholesterol biosynthesisBetacellulin EGF family member mitogen and promoter of beta cell 4 nMdifferentiation. These two functions have been ascribed to differentdomains of the protein Activin A TGF-β family member differentiationfactor, causes ductallike 4 nM cell lines to differentiate intoendocrine pancreas cells Exendin-4 glucagon-like peptide 1 More stableform of GLP-1 (see below) 20 nM agonist Glucagon-like peptide hormone,one of induces glucose production, insulin 20 nM peptide 1 (GLP1) theprotein products from secretion, induces beta cell the glucagon gene;neogenesis, induced beta cell G-protein coupled proliferation receptorligand Hepatocyte Growth Ligand for the c-Met Increases beta cell massin transgenic 10 ng/ml Factor (HGF) receptor animals overexpressing HGF,induces beta cell formation from ductal cell line Niacinamide Member ofvitamin B May affect ADP-ribosylation and/or 10 mM (nicotinamide) familyoxidation state of cell; promotes beta cell differentiation Insulin-likegrowth Peptide hormone Beta cell mitogen 10 nM factor 1 (IGF-I)n-butyrate Histone deacetylase inhibitor (used to 0.5 mM producehepatocytes) to 2.5 mM Retinoic Acid Retinoids differentiation factor 10μM (all trans) Growth Hormone somatotropin/prolactin beta cell mitogen100 ng/ml (Human pituitary family Somatotropin) Placental Lactogensomatotropin/prolactin Increases beta cell mass in transgenic 50 ng/mlfamily animals overexpressing PL, beta cell mitogen during pregnancyVascular Flt-1 and Flk-1 are possible mitogen for endothelial islet 50ng/ml endothelial growth receptors. PDGF/VEGF precursor factor (VEGF)family Insulin-like growth insulin family similar to IGF-1, mitogen, notas 10 nM factor II (IGF-II) strongly implicated in pancreas developmentor function 3-Isobutyl- phosphodiesterase increases insulin secretion100 μM 1-methylxanthine inhibitor, increases cAMP (IBMX) levelswortmannin PI-3 kinase inhibitor may increase insulin secretion 30 nMGastrin Peptide hormone May promote beta cell neogenesis 10 ng/mlCholecystokinin Gut hormone May aid in maturation of islets 5 μM Nervegrowth factor NGF family increases insulin secretion 50 ng/ml (NGF)Epidermal growth EGF family (see transgenic overexpression results in 4nM factor (EGF) betacellulin above) proliferation of beta cellsKeratinocyte growth FGF family member transgenic overexpression resultsin 10 ng/ml factor (KGF), proliferation of beta cells aka FGF7Platelet-derived PDGF family (see VEGF potential precursor mitogen 50ng/ml growth factor above) (PDGF) Regenerating gene Reg family Plays arole in islet regeneration after 100 ng/ml (Reg) pancreas damage Isletneogenesis- Reg family May play a role in islet regeneration 100 ng/mlassociated protein (INGAP)Other ligands or antibodies that bind the same receptors can beconsidered equivalents to any of the receptor ligands referred to inthis disclosure.

Typically, at least two, three, or more than three such factors arecombined in the differentiation cocktail. Human proteins are preferred,but species homologs and variants may also be used. In place of any ofthese factors, the reader may use other ligands that bind the samereceptors or stimulate the same signal transduction pathways, such asreceptor-specific antibody. In addition, other components may beincluded in the medium to neutralize the effect of other factors thatmay be present to drive differentiation down a different pathway.

The efficacy of any of these factors can be determined empirically usinga matrix strategy to arrive at combinations capable of promotingdifferentiation one or more steps down the islet cell pathway. Efficacyis assessed by monitoring emergence of the phenotype of the intendedintermediate or end-stage cell, using the phenotypic markers such asthose listed below. For example, factors believed to induce endocrinepancreas differentiation or proliferation are tested for their abilityto induce Pdx1 expression and subsequently and insulin expression instandard culture conditions.

A fractional factorial design strategy can be used to screen severalcompounds in an efficient manner. Each factor is assigned two levels:for example, the culture matrix would be assigned fibronectin for onelevel and laminin for the second level. In 64 factor combinations (64experiments), it is possible to determine which factors (from a group of15-20) significantly influence differentiation in a statistically robustmanner. Combinations suitable for analysis by this strategy includecyclopamine, TGF family members (TGF-α, Activin A, Activin B, TGFβ1,TGFβ3), Exendin 4, nicotinamide, n-butyrate, DMSO, all-trans retinoicacid, GLP-1, bone morphogenic proteins (BMP-2, BMP-5, BMP-6, BMP-7),insulin-like growth factors (IGF-I, IGF-II), fibroblast growth factor(FGF7, FGF10, bFGF, FGF4), other growth factors (EGF, betacellulin,growth hormone, HGF), other hormones (prolactin, cholecytokinin, gastrin1, placental lactogen), TGF-β family antagonists (Noggin, follistatin,chordin), IBMX, wortmannin, dexamethazone, Reg, INGAP, cAMP or cAMPactivators (forskolin), and extracellular matrix components (laminin,fibronectin). The emerging cell populations are assessed by phenotypicmarkers, and expression patterns are analyzed to determine which factorshave a positive or negative influence on the differentiation pathway.

Directing or Monitoring Differentiation Using Genetically Modified Cells

In optimizing a differentiation or separation strategy, it is sometimeshelpful to use cells that have been genetically altered with a vector inwhich a tissue-specific promoter drives expression of a reporter gene.

Suitable promoters specific for islet progenitors include those drivingtranscription of Pdx1 (NT_(—)009799), Neurogenin 3 (NT_(—)008583),NeuroD1 (NT_(—)005265), Nestin (NT_(—)004858), and Ptf1a-p48(NT_(—)008705). Suitable promoters specific for mature islet cells arethose driving expression of insulin (GenBank Accession NT-009308),glucagon (NT_(—)022154), somatostatin (NT_(—)005962), or pancreaticpolypeptide (NT_(—)010755). A minimal effective sequence of the chosenpromoter (˜0.5 to 5 kB of upstream sequence) is amplified by PCR andspliced into a standard plasmid, adenovirus, lentivirus, or retrovirusvector, in a position that drives expression of a suitable reportergene. Suitable reporter genes either encode a fluorescent molecule (suchas green fluorescent protein or luciferase), encode a detectable enzyme(such as alkaline phosphatase), or produce a cell surface antigen (anyheterologous protein or carbohydrate) that can be detected by antibodyor lectin binding.

Cells transfected with a tissue-specific promoter can be used tooptimize differentiation procedures in the manner already described. Forexample, transfected undifferentiated pPS cells or cells in the earlystage of differentiation can be subject to various differentiationregimes, and then analyzed for the proportion of cells reportingexpression driven by the islet-specific promoter. This provides a rapidread-out of effective differentiation agents, culture environments, andtiming. The optimal procedure can then be used with untransfected cellsto generate high-quality populations of the islet lineage that have anative genotype.

Cells transfected with a tissue-specific promoter can also be used as ameans for accomplishing mechanical sorting. For example, the promotermay drive expression of a drug-resistant phenotype, such as the neomycinresistance gene (conferring resistance to the drug G418) or theblasticidin resistance gene. In this case, once the cells aredifferentiated, they are cultured with the drug to promote outgrowth ofthe desired cell type. Alternatively, the reporter gene may encode afluorescent molecule, or cause expression of a detectable surfaceantigen. In this case, cells of interest are sorted from a population ofdifferentiated cells by cell sorting based on either direct or indirectfluorescence, or by immunoadsorption. A non-replicative, non-integratingvector (such as an adenovirus vector) can be used for transienttransfection during the sorting step, and will dilute out uponsubsequent proliferation of the cell, leaving the cell with a nativegenotype.

The cells can also be genetically altered for the purpose of directlydriving the cells further down the differentiation pathway for isletcells or their progenitors. It is hypothesized that deliberateup-regulation of certain genes normally expressed in the islet cellswill cause less differentiated cells to recruit the genetic expressionprofile appropriate for more mature islet cells. Suitable genes includethose that encode transcription regulators capable of influencingdownstream gene expression. Candidates for pancreatic ontogeny (roughlyin order of their position in the pathway) include Sox17 (GenBankAccession NM_(—)022454), Hlxb9 (NM_(—)005515), Pdx1 (NM_(—)000209),Neurogenin3 (NM_(—)020999), Pax4 (NM_(—)006193) or NeuroD1(NM_(—)002500), Isl1 (NM_(—)002202), Nkx2.2 (NM_(—)002509), and Nkx6.1(NM_(—)006168).

An illustration of this approach is provided in Example 6. Neurogenin 3expression induced by transduction of cells in an islet celldifferentiation paradigm enhances expression in downstream regulatorygenes, and genes encoding islet hormones. The neurogenins are a familyof a neuroD-related bHLH transcription factors: neuronal determinationgenes active in the developing nervous system.

Characteristics of Differentiated Cells

Cells can be characterized according to phenotypic criteria, such asmicroscopic observation of morphological features, detection orquantitation of expressed cell markers, functional criteria measurablein vitro, and behavior upon infusion into a host animal.

Phenotypic Markers

Cells of this invention can be characterized according to whether theyexpress phenotypic markers characteristic of islet cells of variouskinds. Useful markers include those shown in Table 2.

TABLE 2 Phenotypic Markers for Cell Identification SSEA-4 Embryonic stemand germ cells Oct-4 Undifferentiated embryonic pluripotent cellsTelomerase reverse Cells capable of unlimited replication transcriptase(TERT) (e.g., undifferentiated pPS cells) Pdx1 expressed beforepancreatic bud formation in the region of the duodenum that give rise tothe pancreas. Also expressed in mature beta cells Neurogenin 3 (Ngn3)marker of islet precursor cells. Glucagon alpha cells Nkx6.1 beta cellmarker glucokinase beta cell marker Insulin or proinsulin mature betacells Somatostatin delta cells pancreatic polypeptide PP cells isletamyloid polypeptide islet marker (IAPP) Islet-1 islet marker, alsoneural expression Beta-2/NeuroD pan islet cell marker HNF3b endodermmarker HNF4a endoderm marker Sox17 definitive endoderm marker Amylaseexocrine cells HES exocrine pancreas marker nestin potential precursorcell marker Sonic hedgehog signaling molecule, absence is required forpancreas formation CK19 pancreatic duct marker (possible pancreaticprecursor) Glut-2 glucose transporter Patched homologue hedgehogreceptor Smoothened homologue hedgehog receptor

Tissue-specific markers can be detected using any suitable immunologicaltechnique—such as flow immunocytochemistry for cell-surface markers, orimmunohistochemistry (for example, of fixed cells or tissue sections)for intracellular or cell-surface markers. A detailed method for flowcytometry analysis is provided in Gallacher et al., Blood 96:1740, 2000.Expression of a cell-surface antigen is defined as positive if asignificantly detectable amount of antibody will bind to the antigen ina standard immunocytochemistry or flow cytometry assay, optionally afterfixation of the cells, and optionally using a labeled secondary antibodyor other conjugate to amplify labeling. Current sources of specificantibody include the following: Insulin, Sigma Aldrich (12018);glucagon, Sigma Aldrich (G2654); somatostatin, Santa Cruz Biotech(sc-7820); neurogenin 3, Chemicon (AB5684); nestin, BD Transduction Labs(N17220); α-amylase, Sigma Aldrich (A8273); Glut-2, Alpha Diagnostics(GT-22A)

The expression of tissue-specific gene products can also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods. See U.S. Pat. No. 5,843,780 for further details.Sequence data for particular markers listed in this disclosure can beobtained from public databases such as GenBank.

Amplification probe/primer combinations suitable for use inamplification assays include the following: Ins (GenBank NM_(—)000207):primers CAGCCTTTGT GAACCAACACC (SEQ. ID NO:1); CGTTCCCCG CACACTAGGTA(SEQ. ID NO:2); probe CGGCTCACA CCTGGTGGA AGCTC (SEQ. ID NO:3). Nkx6.1(NM_(—)006168): primers CTGGAGGG ACGCACGC (SEQ. ID NO:4); TCCCGTCTTTGTCCMCAAAA (SEQ. ID NO:5); probe TGGCCTGT ACCCCTCATC MGGATCC (SEQ. IDNO:6). Pdx1 (NM_(—)000209): primers TGGCGTTGT TTGTGGCTG (SEQ. ID NO:7);AGGTCCCAA GGTGGAGTGC (SEQ. ID NO:8); probe TGCGCA CATCCCTGCCC TCCTAC.Ngn3 (NM_(—)020999): primers TCTCTATTCT TTTGCGCCGG (SEQ. ID NO:10);CTTGGACAG TGGGCGCAC (SEQ. ID NO:11); probe AGAAAG GATGACGCCTCM CCCTCG(SEQ. ID NO:12). IAPP (NM_(—)000415): primers MGGCAGGA GMTCGCTTGA (SEQ.ID NO:13); TGGTGCMT CTCGGCTCA (SEQ. ID NO:14); probe CCCAGG AGGCGGAGGTTGCA (SEQ. ID NO:15).

HNF3b (NM_(—)021784): primers CCGACTGGAG CAGCTACTATG (SEQ. ID NO:16);TACGTGTTCA TGCCGTTCAT (SEQ. ID NO:17); probe CAGAGCCCGA GGGCTACTCC TCC(SEQ. ID NO:18). p48 (XM_(—)166107): primers CAGGCCCAGA AGGTCATC (SEQ.ID NO:19); GGGAGGGAGG CCATAATC (SEQ. ID NO:20); probe ATCTGCCATCGGGGCACCC (SEQ. ID NO:21). HNF6 (XM_(—)030712): primers AATACCAAAGAGGTGGCGCA (SEQ. ID NO:22); ATGGCCTGTG GGATGCTGT (SEQ. ID NO:23); probeCGTATCACCA CCGAGCTCM GCGC (SEQ. ID NO:24). glucagon (NM_(—)002054):primers GCTGCCAAGG AATTCATTGC (SEQ. ID NO:25); CTTCMCMT GGCGACCTCTTC(SEQ. ID NO:26); probe TGAAAGGCCG AGGAAGGCGA GATT (SEQ. ID NO:27). HNF6(NM_(—)030712): primers GCTGGGGTGA CCTCMTCTA (SEQ. ID NO:28); CAGGMCCTGCATGAGGACT (SEQ. ID NO:29); probe AGTTTCAGAG CCATTGGGCG GTG (SEQ. IDNO:30). NeuroD1 (NM_(—)002500): primers TCACTGCTCA GGACCTACTAACA (SEQ.ID NO:31); GAGGACCTTG GGGCTGAG (SEQ. ID NO:32); probe TACAGCGAGAGTGGGCTGAT GGG (SEQ. ID NO:33). HNF4a (NM_(—)000457): primers GAGATCCATGGTGTTCAAGGA (SEQ. ID NO:34); GTCMGGATG CGTATGGACA (SEQ. ID NO:35); probeCTACATTGTC CCTCGGCACT GCC (SEQ. ID NO:36). Sox17 (NM_(—)022454): primersCAGCAGAATC CAGACCTGCA (SEQ. ID NO:37); GTCAGCGCCT TCCACGACT (SEQ. IDNO:38); probe ACGCCGAGTT GAGCMGATG CTGG (SEQ. ID NO:39). Hlxb9(NM_(—)005515): primers GCCACCTCGC TCATGCTC (SEQ. ID NO:40); CCATTTCATCCGCCGGTTC (SEQ. ID NO:41); probe CCGAGACCCA GGGAAGATTT GGTTCC (SEQ. IDNO:42). Nkx2.2 (NM_(—)002509): primers CGAGGGCCTT CAGTACTCC (SEQ. IDNO:43); TTGTCATTGT CCGGTGACTC (SEQ. ID NO:44); probe ACTCAAGCTCCAAGTCCCCG GAG (SEQ. ID NO:45).

Initial assessment can be done using a marker combination that providesa wide ontogenic profile: for example, Pdx1 for early pancreas cells;Ngn3 for early pancreatic endocrine cells; and insulin for mature betacells. The cells are harvested from the differentiation paradigmsdescribed earlier at regular intervals (say, weekly) to determine thekinetics of differentiation. Cells that test positive for these markerscan then be analyzed for expression of other markers, such as IAPP andNkx6.1. Once the cells are characterized, differentiation factorcombinations and the timing of each step can be optimized.

Certain embodiments of this invention relate to populations in which atleast 2%, 5%, 10%, or more of the cells bear the surface markersreferred to above, either alone or in combination. Endocrine function iscritical to many research and therapeutic applications, in which casepopulations comprising at least 5% of the cells secreting insulin,glucagon, somatostatin, or pancreatic polypeptide are of particularinterest, as are progenitor cells capable of differentiating into suchendocrine-secreting cells. It is a hypothesis of this invention thatinteraction between the alpha, beta, and delta cells may be important inpreventing dedifferentiation and maintaining efficient endocrinesecretion. This invention also includes masses or clusters of cells(perhaps 50-5,000 cells in size), containing two or three of these celltypes, either bound in a matrix of their own making, with a matrixcomponent supplied in culture, or by microencapsulation.

Also desirable are populations with a low residual proportion ofundifferentiated pPS cells. Preferred populations are less than 1%, or0.2% SSEA-4+ve, Oct-4+ve, or positive for expression of endogenoustelomerase reverse transcriptase. Preferred populations also haverelatively low proportions (<5%, 1%, or 0.2%) of certain other celltypes, such as hepatocytes (albumin positive cells), skeletal musclecells (myoD positive), smooth muscle cells (smooth muscle actin), cellswith fibroblast morphology, or neurons (β-tubulin III or NCAM positivecells).

When derived from an established line of pPS cells, the cell populationsand isolated cells of this invention will have the same genome as theline from which they are derived. This means that over and above anykaryotype abnormalities, the chromosomal DNA will be over 90% identicalbetween the pPS cells and the islet cells, which can be inferred if theislet cells are obtained from the undifferentiated line through thecourse of normal mitotic division. Islet cells that have been treated byrecombinant methods to introduce a transgene or knock out an endogenousgene are still considered to have the same genome as the line from whichthey are derived, since all non-manipulated genetic elements arepreserved.

Animal Model Experiments

Of considerable interest for the purposes of islet cells for clinicalapplication is the ability of cell populations to reconstitute the isletsystem of a host animal. Reconstitution can be tested using severalwell-established animal models.

The non-obese diabetic (NOD) mouse carries a genetic defect that resultsin insulitis showing at several weeks of age (Yoshida et al., Rev.Immunogenet. 2:140, 2000). 60-90% of the females develop overt diabetesby 20-30 weeks. The immune-related pathology appears to be similar tothat in human Type I diabetes. Other models of Type I diabetes are micewith transgene and knockout mutations (Wong et al., Immunol. Rev.169:93, 1999). A rat model for spontaneous Type I diabetes was recentlyreported by Lenzen et al. (Diabetologia 44:1189, 2001). Hyperglycemiacan also be induced in mice (>500 mg glucose/dL) by way of a singleintraperitoneal injection of streptozotocin (Soria et al., Diabetes49:157, 2000), or by sequential low doses of streptozotocin (Ito et al.,Environ. Toxicol. Pharmacol. 9:71, 2001). To test the efficacy ofimplanted islet cells, the mice are monitored for return of glucose tonormal levels (<200 mg/dL).

Larger animals provide a good model for following the sequelae ofchronic hyperglycemia. Dogs can be rendered insulin-dependent byremoving the pancreas (J. Endocrinol. 158:49, 2001), or by feedinggalactose (Kador et al., Arch. Opthalmol. 113:352, 1995). There is alsoan inherited model for Type 1 diabetes in keeshond dogs (Am. J. Pathol.105:194, 1981). Early work with a dog model (Banting et al., Can. Med.Assoc. J. 22:141, 1922) resulted in a couple of Canadians making a longocean journey to Stockholm in February of 1925.

By way of illustration, a pilot study can be conducted using pPS derivedislet cells in the following animals: a) non-diabetic nude (T-celldeficient) mice; b) nude mice rendered diabetic by streptozotocintreatment; and c) nude mice in the process of regenerating isletsfollowing partial pancreatectomy. The number of cells transplanted isequivalent to ˜1000-2000 normal human islets, implanted under the kidneycapsule, in the liver, or in the pancreas. For non-diabetic mice, theendpoints of can be assessment of graft survival (histologicalexamination) and determination of insulin production by biochemicalanalysis, RIA, ELISA, and immunohistochemistry. Streptozotocin treatedand partially pancreatectomized animals can also be evaluated forsurvival, metabolic control (blood glucose) and weight gain.

Genetic Modification of Differentiated Cells

The islet precursor cells of this invention have a substantialproliferation capacity. If desired, the replication capacity can befurther enhanced by increasing the level of telomerase reversetranscriptase (TERT) in the cell, either by increasing transcriptionfrom the endogenous gene, or by introducing a transgene. Particularlysuitable is the catalytic component of human telomerase (hTERT),provided in International Patent Application WO 98/14592. Transfectionand expression of telomerase in human cells is described in Bodnar etal., Science 279:349, 1998 and Jiang et al., Nat. Genet. 21:111, 1999.Genetically altered cells can be assessed for hTERT expression byRT-PCR, telomerase activity (TRAP assay), immunocytochemical stainingfor hTERT, or replicative capacity, according to standard methods. Othermethods of immortalizing cells are also contemplated, such astransforming the cells with DNA encoding myc, the SV40 large T antigen,or MOT-2 (U.S. Pat. No. 5,869,243, International Patent Applications WO97/32972 and WO 01/23555).

If desired, the cells of this invention can be prepared or furthertreated to remove undifferentiated cells in vitro, or to safeguardagainst revertants in vivo. One way of depleting undifferentiated stemcells from the population is to transfect the population with a vectorin which an effector gene under control of a promoter that causespreferential expression in undifferentiated cells—such as the TERTpromoter or the OCT-4 promoter. The effector gene may be a reporter toguide cell sorting, such as green fluorescent protein. The effector maybe directly lytic to the cell, encoding, for example, a toxin, or amediator of apoptosis, such as caspase (Shinoura et al., Cancer GeneTher. 7:739, 2000). The effector gene may have the effect of renderingthe cell susceptible to toxic effects of an external agent, such as anantibody or a prodrug. Exemplary is a herpes simplex thymidine kinase(tk) gene, which causes cells in which it is expressed to be susceptibleto ganciclovir (U.S. Ser. No. 60/253,443). Alternatively, the effectorcan cause cell surface expression of a foreign determinant that makesany cells that revert to an undifferentiated phenotype susceptible tonaturally occurring antibody in vivo (U.S. Ser. No. 60/253,357).

Use of Islet Cells in Research and Clinical Therapy

This invention provides a method to produce large numbers of isletprecursor cells, and mature islet cells. These cell populations can beused for a variety of important research, development, and commercialpurposes.

The cells of this invention can be used to prepare a cDNA libraryrelatively uncontaminated with cDNA preferentially expressed in cellsfrom other lineages. The differentiated cells of this invention can alsobe used to prepare monoclonal or polyclonal antibodies that are specificfor markers of islet precursors and their derivatives, according tostandard methods.

Of particular interest are use of the compositions of this invention fordrug development and clinical therapy.

Drug Screening

Islet cells of this invention can be used to screen for factors (such assolvents, small molecule drugs, peptides, polynucleotides) orenvironmental conditions (such as culture conditions or manipulation)that affect the characteristics of islet precursor cells and theirvarious progeny.

A prime example is the use of islet cell clusters or homogeneous betacell preparations for the effect of small molecule drugs that have thepotential to up- or down-regulate insulin synthesis or secretion. Thecells are combined with the test compound, and then monitored for changein expression or secretion rate, for example by RT-PCR or immunoassay ofthe culture medium.

Other screening methods of this invention relate to the testing ofpharmaceutical compounds for a potential effect on islet cell growth,development, or toxicity. This type of screening is appropriate not onlywhen the compound is designed to have a pharmacological effect on isletcells, but also to test for islet-related side-effects of compoundsdesigned for a primary pharmacological effect elsewhere.

In a third example, pPS cells (undifferentiated or differentiated) areused to screen factors that promote maturation into islet cells, orpromote proliferation and maintenance of islet cells in long-termculture. For example, candidate differentiation factors or maturationfactors are tested by adding them to cells in different wells, and thendetermining any phenotypic change that results, according to desirablecriteria for further culture and use of the cells. This can lead toimproved derivation and culture methods for not only pPS derived islets,but also for islet cells and their progenitors isolated from pancreas.

The reader is referred generally to the standard textbook “In vitroMethods in Pharmaceutical Research”, Academic Press, 1997, and U.S. Pat.No. 5,030,015. Assessment of the activity of candidate pharmaceuticalcompounds generally involves combining the differentiated cells of thisinvention with the candidate compound, either alone or in combinationwith other drugs. The investigator determines any change in themorphology, marker phenotype, or functional activity of the cells thatis attributable to the compound (compared with untreated cells or cellstreated with an inert compound), and then correlates the effect of thecompound with the observed change.

Cytotoxicity can be determined in the first instance by the effect oncell viability, survival, morphology, and the expression of certainmarkers and receptors. Effects of a drug on chromosomal DNA can bedetermined by measuring DNA synthesis or repair. [³H]-thymidine or BrdUincorporation, especially at unscheduled times in the cell cycle, orabove the level required for cell replication, is consistent with a drugeffect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread. The reader isreferred to A. Vickers (pp 375-410 in “In vitro Methods inPharmaceutical Research,” Academic Press, 1997) for further elaboration.

Reconstitution of Islet Function

This invention also provides for the use of islet precursor cells ortheir derivatives to restore islet function in a patient in need of suchtherapy. Any condition relating to inadequate production of a pancreaticendocrine (insulin, glucagon, or somatostatin), or the inability toproperly regulate secretion may be considered for treatment with cellsprepared according to this invention, as appropriate. Of especialinterest is the treatment of Type I (insulin-dependent) diabetesmellitus.

Patients are chosen for treatment based on confirmed long-termdependence on administration of exogenous insulin, and acceptable riskprofile. The patient receives approximately 10,000 islet equivalents perkg body weight. To overcome an allotype mismatch, the patient is startedbefore surgery with anti-rejection drugs such as FK506 and rapamycin(orally) and daclizumab (intravenously). The islet cells are infusedthrough a catheter in the portal vein. The patient is then subjected toabdominal ultrasound and blood tests to determine liver function. Dailyinsulin requirement is tracked, and the patient is given a secondtransplant if required. Follow-up monitoring includes frequent bloodtests for drug levels, immune function, general health status, andwhether the patient remains insulin independent.

General approaches to the management of the diabetic patient areprovided in standard textbooks, such as the Textbook of InternalMedicine, 3^(rd) Edition, by W. N. Kelley ed., Lippincott-Raven, 1997;and in specialized references such as Diabetes Mellitus: A Fundamentaland Clinical Text 2nd Edition, by D. Leroith ed., Lippincott Williams &Wilkins 2000; Diabetes (Atlas of Clinical Endocrinology Vol. 2) by C. R.Kahn et al. eds., Blackwell Science 1999; and Medical Management of TypeI Diabetes 3^(rd) Edition, McGraw Hill 1998. Use of islet cells for thetreatment of Type I diabetes is discussed at length in CellularInter-Relationships in the Pancreas: Implications for IsletTransplantation, by L. Rosenberg et al., Chapman & Hall 1999; and FetalIslet Transplantation, by C. M. Peterson et al. eds., Kluwer 1995.

As always, the ultimate responsibility for patient selection, the modeof administration, and dosage of pancreatic endocrine cells is theresponsibility of the managing clinician.

For purposes of commercial distribution, islet cells of this inventionare typically supplied in the form of a pharmaceutical composition,comprising an isotonic excipient prepared under sufficiently sterileconditions for human administration. This invention also includes setsof cells that exist at any time during their manufacture, distribution,or use. The cell sets comprise any combination of two or more cellpopulations described in this disclosure, exemplified but not limited toa type of differentiated pPS-derived cell (islet cells, theirprecursors, subtypes, and so on), in combination with undifferentiatedpPS cells or other differentiated cell types, sometimes sharing the samegenome. Each cell type in the set may be packaged together, or inseparate containers in the same facility, or at different locations,under control of the same entity or different entities sharing abusiness relationship.

For general principles in medicinal formulation of cell compositions,the reader is referred to Cell Therapy: Stem Cell Transplantation, GeneTherapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds,Cambridge University Press, 1996. The composition is optionally packagedin a suitable container with written instructions for a desired purpose,such as the treatment of diabetes.

Devices

The cells of this invention can also be used as the functional componentin a mechanical device designed to produce one or more of the endocrinepolypeptides of pancreatic islet cells.

In its simplest form, the device contains the pPS derived islet cellsbehind a semipermeable membrane that prevents passage of the cellpopulation, retaining them in the device, but permits passage ofinsulin, glucagon, or somatostatin secreted by the cell population. Thisincludes islet cells that are microencapsulated, typically in the formof cell clusters to permit the cell interaction that inhibitsdedifferentiation. For example, U.S. Pat. No. 4,391,909 describe isletcells encapsulated in a spheroid semipermeable membrane made up ofpolysaccharide polymers>3,000 mol. wt. that are cross-linked so that itis permeable to proteins the size of insulin, but impermeable tomolecules over 100,000 mol. wt. U.S. Pat. No. 6,023,009 describes isletcells encapsulated in a semipermeable membrane made of agarose andagaropectin. Microcapsules of this nature are adapted for administrationinto the body cavity of a diabetic patient, and are thought to havecertain advantages in reducing histocompatibility problems orsusceptibility to bacteria.

More elaborate devices are also contemplated, either for implantationinto diabetic patients, or for extracorporeal therapy. U.S. Pat. No.4,378,016 describes an artificial endocrine gland containing anextracorporeal segment, a subcutaneous segment, and a replaceableenvelope containing the hormone-producing cells. U.S. Pat. No. 5,674,289describes a bioartificial pancreas having an islet chamber, separated bya semipermeable membrane to one or more vascularizing chambers open tosurrounding tissue. Useful devices typically have a chamber adapted tocontain the islet cells, and a chamber separated from the islet cells bya semipermeable membrane which collects the secreted proteins from theislet cells, and which may also permit signaling back to the isletcells, for example, of the circulating glucose level.

The following examples are provided as further non-limitingillustrations of particular embodiments of the invention.

EXAMPLES Example 1 Feeder-Free Propagation of Embryonic Stem Cells

Established lines of undifferentiated human embryonic stem (hES) cellswere maintained in a culture environment essentially free of feedercells.

Conditioned medium prepared in advance using primary mouse embryonicfibroblasts (mEF) isolated according to standard procedures (WO01/51616). Fibroblasts were harvested from T 50 flasks by washing oncewith Ca⁺⁺/Mg⁺⁺ free PBS and incubating in 1.5-2 mL trypsin/EDTA (Gibco)for ˜5 min. After the fibroblasts detached from the flask, they werecollected in mEF medium (DMEM+10% FBS). The cells were irradiated at4000 rad, counted, and seeded at ˜55,000 cells cm⁻² in mEF medium. Afterat least 4 h, the medium were exchanged with SR containing ES medium(80% knockout DMEM (Gibco BRL, Rockville Md.), 20% knockout serumreplacement (Gibco), 1% non-essential amino acids (Gibco), 1 mML-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Sigma, St. Louis, Mo.),supplemented with 4 ng/mL recombinant human basic fibroblast growthfactor (bFGF; Gibco). About 0.3-0.4 mL of medium was conditioned per cm²of plate surface area. Before addition to the hES cultures, theconditioned medium was supplemented with another 4 ng/mL of human bFGF.

Plates for culturing the hES cells were coated with Matrigel®(Becton-Dickinson, Bedford Mass.) by diluting stock solution ˜1:30 incold KO DMEM, dispensing at 0.75-1.0 mL per 9.6 cm² well, and incubatingfor 4 h at room temp or overnight at 4° C.

hES cultures were passaged by incubation in ˜200 U/mL collagenase IV for˜5-10 min at 37° C. Cells were harvested by removing individual coloniesup with a Pipetman™ under a microscope or scraping, followed by gentledissociation into small clusters in conditioned medium, and then seededonto Matrigel® coated plates. About one week after seeding, the culturesbecame confluent and could be passaged. Cultures maintained under theseconditions for over 180 days continued to display ES-like morphology.SSEA-4, Tra-1-60, Tra-1-81, and alkaline phosphatase were expressed bythe hES colonies, as assessed by immunocytochemistry, but not by thedifferentiated cells in between the colonies.

Expression of the undifferentiated hES cell markers was assayed byreverse-transcriptase PCR amplification. The transcription factor Oct-4is normally expressed in the undifferentiated hES cells and isdown-regulated upon differentiation. Cells maintained on Matrigel® inconditioned medium for 21 days expressed hTERT and Oct-4. Telomeraseactivity was measured by TRAP assay (Kim et al., Science 266:2011, 1997;Weinrich et al., Nature Genetics 17:498, 1997). Cells maintained in thefeeder-free culture were telomerase positive.

Pluripotency of undifferentiated cells cultured without feeders wasdetermined by differentiating the cells through the formation ofembryoid bodies. Confluent monolayer cultures of hES cells wereharvested by incubating in 1 mg/mL collagenase for 5-20 min, anddissociated into clusters. They were then plated in non-adherent cellculture plates (Costar) in a medium composed of 80% KO DMEM (Gibco) and20% non-heat-inactivated FBS (Hyclone), supplemented with 1%non-essential amino acids, 1 mM glutamine, 0.1 mM p-mercaptoethanol. Theembryoid bodies were fed every other day by the addition of 2 mL ofmedium per well. After 4-8 days in suspension, they were then culturedon poly-ornithine coated plates for about 7 days.

Immunocytochemistry showed staining patterns consistent with cells ofthe neuron and cardiomyocyte lineages, and cells staining forα-fetoprotein, a marker of endoderm lineage. The undifferentiated cellswere also tested for their ability to form teratomas by intramuscularinjection into SCID mice. Resulting tumors were excised after 78-84days. Cell types from all three germ layers were identified byhistological analysis.

Example 2 Derivation of Hepatocytes from hES Cells

Undifferentiated hES cells were differentiated into cells havingcharacteristics of human hepatocytes using a strategy to initiate aglobal differentiation process using DMSO in a subconfluent culture. Thecells are then induced to form hepatocyte-like cells by the addition ofNa-butyrate.

Briefly, the hES cells were maintained in undifferentiated cultureconditions for 2-3 days after splitting. At this time, the cells were 50to 60% confluent and the medium was exchanged with unconditioned SRmedium containing 1% DMSO (Step II). The cultures were fed daily with SRmedium for 4 days and then exchanged into unconditioned SR mediumcontaining both 1% DMSO and 2.5% Na-butyrate, with which they were feddaily for 6 days (Step III). They were then replated onto collagen, andcultured in a hepatocyte maturation medium containing a cocktail ofhepatocyte-friendly growth factors (Step IV). The procedure issummarized in Table 3.

TABLE 3 Hepatocyte Differentiation Protocol Step IV Step I Step IIIHepatocyte Undifferentiated Step II Hepatocyte maturation cellsPre-differentiation differentiation (Groups 7-9 only; (until confluent)(4 days) (6 days) 4 days) Feeder-free 20% SR medium + 1% 20% SR medium +1% HCM + 30 ng/mL conditions DMSO DMSO + 2.5 mM hEGF + 10 ng/mL butyrateTGF-α + 30 ng/mL HGF + 2.5 mM butyrate

FIG. 1 shows the hepatocyte-like cells that were obtained. Left column:10× magnification; Right column: 40× magnification. By 4 days in thepresence of butyrate, more than 80% of cells in the culture are large indiameter, containing large nuclei and granular cytoplasm (Row A). After5 days in SR medium, the cells were switched to HCM. Two days later,many cells are multinucleated, and have a large polygonal shape (Row B).By 4 days in HCM, multinucleated polygonal cells are common, and have adarker cytosol (Row C), by which criteria they resemble freshly isolatedhuman adult hepatocytes (Row D) or fetal hepatocytes (Row E).

Example 3 Obtaining Insulin-Secreting Cells from Early Cells in theHepatocyte Pathway

Insulin-secreting cells were derived from hES cells of the H9 line usinga modification of the hepatocyte differentiation protocol described inthe last Example.

Briefly, in Step I, hES cells were grown to confluence after their lastpassage over a 7-8 day period using SR medium conditioned by mouseembryonic fibroblasts as described in Example 1. In Step II, the mediumwas exchanged with unconditioned SR medium containing 1% DMSO, to which10 μM cyclopamine was added, and the cells were cultured for 4 days. InStep III, the cells were cultured for 11 days in RPMI1640 medium withB27 supplement, containing 0.5 mM butyrate (5-fold lower than thehepatocyte protocol), 10 μM cyclopamine, 4 nM activin A and 10 mMnicotinamide. The cells were then harvested, fixed, and stained forinsulin expression (using the Sigma mouse monoclonal anti-insulinantibody at a dilution of 1:500) and counterstained with DAPI to detectnuclei. In Step IV, the cells are matured RPMI1640/B27 by culturing for11 days in RPMI supplemented with B27, and containing possible isletdifferentiation factors.

FIG. 2 shows the results in which the cells were cultured in Step IVwith 4 nM activin A, 4 nM betacellulin, 10 nM IGF-1, and 10 mMnicotinamide. The round circles in the upper panel represent the DAPIblue-stained nuclei. The diffuse staining centered in the middle of thefield is red fluorescent staining, representing insulin synthesized andfixed within the cell. It was estimated that about 1% of the cells inthe well were expressing insulin. No insulin staining was observed inother cell clusters on the same slide or in the isotype control.

Example 4 Entering the Islet Cell Pathway by Optimizing Formation of GutEndoderm

This example provides an illustration of the stepwise induction ofislets from pPS cells, mimicking the normal development that occurs inutero.

The H7 and H9 line of hES cells were grown to confluence using standardfeeder free conditions, which served as starting material for theexperiment. To initiate differentiation, the medium was changed to RPMI1640 supplemented with B27 (Invitrogen) and one of the followingalternatives:

-   -   no further additives (medium control)    -   4 nM Activin A (R&D systems)    -   0.5 mM sodium butyrate (Sigma)    -   both Activin A and butyrate together.        The medium was exchanged daily (with the exception of one day)        for a total of 8 treatment days.

At the end of this period, the medium was removed, the cells were rinsedwith PBS, and a cell lysate was prepared by the addition of 400 μL ofRTL lysis buffer (Qiagen). RNA was prepared using the Qiagen RNAeasyMini kit according to the manufacturer's instructions. The RNA wastreated with DNAse, and random primed first strand cDNA was preparedusing the Invitrogen Preamplification First Strand cDNA kit followingmanufacturer's instructions. Standard real-time RT-PCR (Taqman) wascarried out on each cDNA sample using primer and probe sets for Sox17,HNF1alpha, and HNF3beta. To normalize expression levels, parallel Taqmanassay was run using the probe and primer set for cycophilin (AppliedBiosystems). The relative abundance of each sample compared to apancreas standard was calculated using the delta delta Ct method(Applied Biosystems).

FIG. 3 shows expression levels of the markers for gut endoderm. Resultsare standardized to the expression level in human fetal pancreas. Alsoshown for comparison is the level of expression in undifferentiated hEScells (the starting cell line). The three markers were induced mosteffectively by the combination of both Activin A and sodium butyrate.Sox17 was expressed at high levels (2.3 relative expression) inundifferentiated H9 cells, but was induced to 32 relative expressionlevel with the two additives (more than 100-fold increase). In H7 cells,the level of induction was more dramatic, going from 0.007 times fetalpancreas to 79 times fetal pancreas (more than 10.000-fold increase).HNF3beta increased from less than 0.5 relative expression to 5.1 or 6.3(more than 10-fold increase). The levels of HNF4alpha increased fromless than 0.1 relative expression level to over 1.0 (more than 10-foldincrease).

The robust induction of these three markers confirms that these hESderived cells have key characteristics of gut endoderm. These cells arenext subcultured and treated with factors that induce pancreasformation, monitored by induction of the key pancreas marker Pdx1.

Example 5 Obtaining Islet Cells by Using Additives in Long-TermAggregate Culture

The H7 line of hES cells was grown in an undifferentiated form in mEFmedium as in Example 1. They were then grown in suspension culture(forming embryoid bodies) in a blended medium consisting of mEFconditioned medium and DFB+ at 1:1. DFB+ is DMEM/F12 medium supplementedwith B27 additive (1×), insulin (25 μg/ml), progesterone (6.3 ng/ml),putrescine (10 μg/ml), selenium (in the form of selenite, 100 ng/ml),transferrin (50 μg/ml), and the thyroid hormone receptor ligand T3 (40ng/ml). The next day, 10 μM all-trans retinoic acid was added. On thethird day, the medium was changed to DFB+ with 10 μM all trans retinoicacid, then fed every other day for 7 days (Stage 1).

To initiate Stage 2, all-trans retinoic acid was removed and replacedwith medium containing Noggin (200 ng/ml), EGF (20 ng/ml) and bFGF (2ng/ml). The cells were fed every other day for 14 days.

For Stage 3, Noggin, EGF and bFGF were withdrawn, and the cells werecultured in DFB+containing nicotinamide (10 mM). The medium was changedevery other day for 5 days. The cells were then plated on coated chamberslides for one day, fixed and stained with antibodies against thec-peptide of insulin, or somatostatin. c-Peptide is a component ofproinsulin that is removed before secretion. It is useful indistinguishing insulin synthesized from the cell from insulin present asa component of the medium.

FIG. 4 shows the results. The top and middle panels show staining forinsulin c-peptide at low and high magnification, indicating a cluster ofmature pancreatic beta cells. The bottom panel shows staining forsomatostatin, a marker of islet delta cells. Taqman real-time RT-PCRconfirmed that insulin and glucagon are expressed by these cells at themRNA level.

Example 6 Driving the End-Stage Islet Pathway by Neurogenin 3 GeneExpression

This experiment demonstrates the effectiveness of overexpressing anislet related gene in recruiting a gene expression profilecharacteristic of the islet cell lineage during Stage III ofdifferentiation.

An adenovirus vector was constructed containing the transcriptionregulator gene Neurogenin 3 (Ngn3) under control of a constitutivepromoter. AdNgn3 is a replication deficient, E1- and E3-deletedrecombinant adenovirus 5 based vector constructed using the AdMAX™vector construction kit (Microbix Biosystems, Toronto). Ngn3 CDNA 645 bp(GenBank accession # NM_(—)020999) was cloned into pDC515 shuttle vectordownstream of mCMV (Murine Cytomegalovirus Immediate Early Gene)promoter. A natural sequence TAGAAAGG immediately upstream of ATG startcodon was replaced with the consensus Kozak sequence GCCACC. The shuttleplasmid containing the Ngn3 insert and the Ad genomic plasmid (AdMAXplasmid) were cotransfected into E1-expressing 293 cells for homologousrecombination to occur. Plaques were isolated, the viral stock wasamplified in 293 cells, and then purified by CsCl density gradientcentrifugation. Viral particle concentration was determined by measuringoptical density at A₂₆₀, and the infectious titer was determined bystandard plaque assay.

Human ES cells of the H7 line were suspended and cultured in blendedmedium as in Example 5. All-trans retinoid acid (10 μM) was added to themedium the next day. Seven days later, the EBs were plated on Matrigel®in DFB+ medium plus EGF (20 ng/ml) and bFGF (2 ng/ml). After five daysin culture, cells were infected with the AdNgn3 vector at MOI of 50.AdGFP (an equivalent vector containing the gene for green fluorescentprotein, not expected to direct differentiation) was used as a negativecontrol. AdNgn3 or AdGFP virus at an MOI of 50 was applied directly tocells in 6-well plates in 1 mL of DFB+ medium containing EGF (20 ng/ml)for 4 h, then additional 2 mL of the same medium was added to each well.The medium was replenished every other day thereafter. Cells wereharvested 2 days or 8 days after the infection, and RNA was isolated foranalysis by real-time RT-PCR as in Example 4. Cells plated on chamberslides for immunohistochemistry were fixed in 4% paraformaldehyde,blocked, and stained with antibody against glucagon (1:1000), followedby FITC conjugated goat anti-mouse IgG (1:500).

FIG. 5 shows immunocytochemical staining of the Neurogenin 3 transducedcells 9 days after transduction. This plated embryoid body shows asubstantial level of antibody-detectable glucagon expression.

FIGS. 6(A) and (B) shows mRNA expression levels in the Neurogenin 3transduced cells (solid bars), compared with the negative control(hatched bars). Real-time RT-PCR data is normalized to expression levelsin human fetal pancreas.

It was found that several genes that are downstream of Neurogenin 3 werespecifically up-regulated in the transduced cells, compared withcontrols. NeuroD1 and Nkx2.2 were both substantially upregulated by day2; Nkx6.1 was upregulated by day 8. Insulin and glucagon expression werealso substantially up-regulated by day 8, indicating that the genetransfection strategy substantially improves maturation to cells makingthe clinically important end-stage products of islet cells.

The skilled reader will appreciate that the invention can be modified asa matter of routine optimization, without departing from the spirit ofthe invention, or the scope of the appended claims.

1. A method of screening a compound for its effect on a primatepancreatic islet cell or a pancreatic islet cell precursor comprising a)isolating a population of primate pancreatic islet cells or pancreaticislet cell precursors from a cell culture comprising primate pluripotentstem cells (pPS); b) contacting the isolated population of primatepancreatic islet cells or pancreatic islet cell precursors with thecompound; and c) determining any affect from the compound to theisolated population of primate pancreatic islet cells or pancreaticislet cell precursors from b).
 2. The method of claim one wherein theprimate pancreatic islet cells or pancreatic islet cell precursors arehuman cells.
 3. The method of claim one comprising determining if thecompound is toxic to the primate pancreatic islet cells or pancreaticislet cell precursors.
 4. The method of claim one comprising determiningif the compound affects insulin expression or insulin secretion.
 5. Themethod of claim one comprising determining if the compound affectsgrowth of primate pancreatic islet cells or pancreatic islet cellprecursors.
 6. A cell culture comprising a) a first population of cellscomprising primate pluripotent stem cells (pPS); b) a second populationof cells comprising cells that express Sox17, HNF3 and HNF4; and c)Activin A at a concentration suitable to induce expression of Sox17,HNF3 and HNF4 at level that is higher in the second population of cellscompared to the first population of cells.
 7. The cell culture of claim6, wherein the primate pluripotent stem cells (pPS) are human cells. 8.The cell culture of claim 6, further comprising butyrate.
 9. The cellculture of claim 6, wherein the second population of cells comprises gutendodermal cells.